Trifunctional and completely clearable specific targeting agents and methods thereof

ABSTRACT

The invention provides a novel trifunctional targeting construct and related compositions and methods that are useful in therapeutic, diagnostic (including imaging) of various biological and/or pathological conditions and diseases such as cancers and diabetes. The trifunctional targeting construct of the invention provides enhanced clearing step and reduced non-specific background via complete clearance of undesired antibody conjugates.

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/811,676, filed Apr. 12, 2013, the entire content of which isincorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with Government support under Grant No. DK094199awarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to therapeutics and diagnostics ofbiological and/or pathological conditions. More particularly, theinvention relates to a unique trifunctional targeting construct fortargeted delivery of therapeutic or diagnostic agents, and relatedcompositions and methods of use and preparation. The compositions andmethods of the invention are useful in treatment and/or diagnosis(including imaging) of various conditions and diseases such as cancersand pancreatic islets in connection with diabetes.

BACKGROUND OF THE INVENTION

Targeted drug delivery is of critical importance in ensuring safety andefficacy of therapeutic and diagnostic agents. In cancer treatment anddiagnosis, for example, targeted delivery directly to the tumor siteallows more effective dosing at the tumor sites than systemic delivery,therefore increasing drug effectiveness while reducing side effects.Diagnostic or therapeutic agents conjugated to targeting moieties suchas antibodies or antibody fragments, cell- or tissue-specific peptides,hormones and other receptor binding molecules have previously beenstudied. (See, e.g., U.S. Pat. Nos. 3,927,193, 4,331,647, 4,348,376,4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and4,818,709).

A significant challenge for direct targeting methods, such as throughantibody vectors, is that a relatively small fraction of the conjugateactually binds to the target site, while the majority of the antibodyconjugate molecules remain in circulation. Such antibody-agent conjugatemolecules often negatively affect the functionality of the active agentand cause undesirable marrow toxicity or other systemic side effects.Besides limiting dosing, the circulating antibody-agent conjugatemolecules also increase background noise and reduce resolution in adiagnostic application. In islet imaging, because islets constitute only1-2% of the pancreatic mass, additional difficulty appears within theorgan and highly specific delivery is more crucial. (See, e.g., Liu, etal. 2011 Mol. Pharmaceutics 8, 767-773; Liu, et al. 2012 NuclearMedicine and Biology 39, 645-651.)

To increase the target to background ratios, pretargeting methods andclearing agents have been studied. An example of pretargeting method isa (strept)avidin-biotin system. Another example is the use of thebispecific antibody-hapten recognition system, which uses a radiolabeledhapten and a bispecific antibody in place of (strept)avidin and biotin.(See, e.g., Barbet, et al. 1999 Cancer Biother. Radiopharm. 14:153-166;Karacay, et al. 2000 Bioconj. Chem. 11: 842-854; Gautherot, et al. 2000J. Nucl. Med. 41:480-487; Lubic, et al. 2001 J. Nucl. Med. 42:670-678;Gestin, et al. 2001 J. Nucl. Med. 42:146-153.) However, the affinity ofan antibody for its hapten, particularly for a monovalent one, is ordersof magnitude lower than that of (strept)avidin for biotin.

Existing targeting and clearance systems, however, suffer from lowclearance efficiency as well as nonspecific background, which remain asmajor hurdles to creating an effective targeted delivery system. A novelpretargeting approach is especially desired that has enhanced clearingeffectiveness and reduced non-specific background.

SUMMARY OF THE INVENTION

The invention provides a unique trifunctional targeting construct andrelated compositions and methods that are useful in therapeutics anddiagnosis (including imaging) of various biological and/or pathologicalconditions and diseases. The trifunctional targeting construct of theinvention enables a much enhanced clearing mechanism and significantlyreduced non-specific background via the completely clearable antibodyconstruct.

In one aspect, the invention generally relates to a conjugate compound.The compound includes: a targeting moiety capable of selectively bindingto a primary and specific target site or to a substance produced by orassociated with the target site; a first conjugate moiety comprising twocovalently linked ligand groups, a first ligand group and a secondligand group; and a covalent linkage between the targeting moiety andthe first conjugate to form a second-order conjugate compound.

In another aspect, the invention generally relates to a kit fordelivering a diagnostic or therapeutic agent to a target site. The kitincludes: (a) a compound that include: a targeting moiety capable ofselectively binding to a primary and specific target site or to asubstance produced by or associated with the target site, a firstconjugate comprising two covalently linked ligand groups, a first ligandgroup and a second ligand group, and a covalent linkage between thetargeting moiety and the first conjugate; (b) a clearing agentcomprising a group capable of selective binding to the second ligandgroup; and (c) an effector conjugate, wherein the effector conjugatecomprises an effecting group covalently linked to a group capable ofselective binding to the first ligand group.

In yet another aspect, the invention generally relates to a method fortargeted delivery of an agent to a target site in a mammal. The methodincludes: (a) administering to the mammal a first compound. The firstcompound includes: a targeting moiety capable of selectively binding toa primary and specific target site or to a substance produced by orassociated with the target site, a first conjugate comprising twocovalently linked ligand groups, a first ligand group and a secondligand group, and a second conjugate formed by a covalently linkagebetween the targeting moiety and the first conjugate; (b) administeringto the mammal a clearing agent comprising a group capable of selectivebinding to the second ligand; and (c) administering to the mammal aneffector conjugate, wherein the effector conjugate comprises an agentcovalently linked to a group capable of selective binding to the firstligand group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the different approaches between thepresent invention (a) and the prior art (b).

FIG. 2 schematically illustrates an exemplary chemical modification (A)of an antibody by both a biotin and a MORF that may produce a mixture ofdesired clearable product, undesired non-clearable product, andnon-interfering unmodified and partially unmodified antibody molecules;and a new design (B) to modify the antibody by SFB-derivatized(biotin-MORF) that produces only the desired clearable antibody togetherwith the non-interfering unmodified and partially unmodified antibodymolecules. SANH and SFB are the paired agents in the commercialHydralink approach to link two groups together. MORF is a DNA analogueof morpholino oligomer that binds to its complement, cMORF (thepretargeting effector).

FIG. 3 shows a scheme for constructing a completely clearable(MORF-biotin)-Ab and evaluating the avidin clearance effect.

FIG. 4 shows (A) absorbance of the reaction mixture of 15 μL ofHABA-avidin complex and increasing amount (0.889 μg/μL) of(NH₂-biotin)-MORF in a reaction buffer, and (B) HPLC traces at UV 265 nmof a mixture containing the (NH₂-biotin)-MORF, an internal standard ofiodohippuric acid, and increasing amounts of streptavidin (SA).

FIG. 5 shows HPLC traces of ^(99m)Tc labeled (NH₂-biotin)-MORF (A)before and after adding excessive cold cMORF or (B) before and afteradding excessive SA. (C) shows the radioactivity uptakes of ^(99m)Tclabeled (NH₂-biotin)-MORF on streptavidin beads along with the negativecontrols of the biotin-free ^(99m)Tc-MORF and the avidin-saturated(^(99m)Tc-biotin)-MORF.

FIG. 6 shows radioactivity levels of avidin-bound (solid circle) andnon-avidin-bound (solid triangle) (A) (^(99m)Tc-biotin)-MORF and (B)(biotin-CC49)-MORF/cMORF-^(99m)Tc in blood, liver, whole body, andkidneys over time.

FIG. 7. Clearance efficiency for different pretargeting intervals in apretargeting procedure using biotin-CC49-MORF, avidin, and^(99m)Tc-cMORF. The clearance efficiency is defined as (1—the ratio ofblood level with/without avidin).

FIG. 8. The blood radioactivity levels after a procedure includinginjection of the biotin-Ab-MORF, different number of injections ofavidin, and the injection of the raidolabeled cMORF. The time ofeuthanization of the mice was at 4 days after initial injection of thepretargeting antibody and 3 h after injection of radiolabeled cMORF (seeMethods).

FIG. 9. The structures of (NH₂-biotin)-MORF and native MORF.

FIG. 10. A scheme describing the MAG₃ conjugation to the MORFs andpurification for ^(99m)Tc labeling.

FIG. 11. (A) The OD values of the reaction mixture of 15 μL ofHABA-avidin with an increasing amount of (NH₂-biotin)-MORF (0.798 μg/4).(B) The HPLC traces at UV 265 nm of the (NH₂-biotin)-MORF mixed with aninternal standard of iodohippuric acid after adding an increasing amountof streptavidin (SA).

FIG. 12. The HPLC traces of native MORF, (NH₂-biotin)-MORF, and their1:1 mixture after NHS-MAG₃ conjugation and radiolabeling (top row). Alsoshown are the peaks after addition of excess cMORF at a molar ratio ofcMORF/MORF=55 (second row) and excess SA at a molar ratio of SA/MORF=10(bottom row).

FIG. 13. The relative reactivity of (NH₂-biotin)-MORF to that ofnative-MORF.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a unique targeting construct, and relatedcompositions and methods of use and preparation, which are useful intreatment and diagnosis (including imaging) of various biological and/orpathological conditions and diseases such as cancers and diabetes. Thetrifunctional targeting construct of the invention ensures minimizationof the undesirable by-products while significantly reduces non-specificbackground via complete clearance of undesired antibody conjugates.

Monoclonal antibodies have been investigated as targeted therapeutic andimaging agents for many years. The recent exciting success inhematological cancer therapy highlights the importance of the “magicbullet” concept. However, compared to the hematological cancers, solidtumor therapy would be more challenging because of its loweraccessibility. Increasing the toxicity or the dose of the “warhead”would address the required toxic effect for tumor therapy, but thetoxicity to normal tissues would be elevated at the same time. Asolution is to clear the circulating antibody out of normal organs afterthe tumor accumulation essentially completes. In addition for therapy,the clearance concept may also be important for tumor imaging as well incertain circumstances, for example, using antibody to image the islet ofLangerhans. (Liu, et al. 2012 Nucl. Med. Biol. 39, 645-651; Liu, et al.2011 Mol. Pharmaceutics 8, 767-773.) Because islets constitute only 1-2%of the pancreas mass and the current nuclear imaging technologies cannotdifferentiate islets from non-islet pancreatic tissues, reduction of thenon-specific binding in the exocrine tissues is critical.

Currently, there are two clearing mechanisms in the literature. Thesecondary antibody mechanism takes advantage of the big size of theaggregate formed between the secondary antibody and the pretargetingantibody to remove the latter from the circulation by thereticuloendothelial (RE) cells. (Goodwin, et al. 1988 J. Nucl. Med. 29,226-234; Goodwin, et al. 1994 Cancer Res. 54, 5937-5946.) The saccharidemechanism employs the clearing agents bearing saccharide groups, forexample, avidin, galactosylated anti-antibodies against the pretargetingantibody, and galactosylated and biotinylated HSA. The complexes formedbetween antibody and clearing agent are removed by an asialoglycoproteinreceptor. Both mechanisms traffic the circulating pretargeting moleculesinto liver. However, although the concept was considered extensively forits application in tumor pretargeting, few efforts were invested tounderstand the in vivo interaction between the antibody and the clearingagent. (Yao, et al. 1995 J. Nucl. Med. 36, 837-841; Wang, et al. 2001Bioconjug. Chem. 12, 807-816; Karacay, et al. 1997 Bioconjug. Chem. 8,585-594; Sharkey, et al. 1997 Bioconjug. Chem. 8, 595-604; Axworthy, etal. 2000 Proc. Natl. Acad. Sci. U.S.A. 97, 1802-1807; Mirallie, et al.2005 Eur. J. Nucl. Med. Mol. Imaging. 32, 901-909; Liu, et al. 2010Cancer Biother. Radiopharm. 25, 757-762; Ashwell, et al. 1974 Adv.Enzymol. 41, 99-128; Ong, et al. 1991 Cancer Res. 51, 1619-1626;Kobayashi, et al. 1995 Eur. J. Cancer 31, 1689-1696.)

The current investigation employs a model pretargeting system toexemplify an investigation into the in vivo chemistry between a clearingagent and a biotinylated IgG antibody. In this system, the antibody CC49as the pretargeting agent is conjugated concomitantly with a morpholinophosphorodiamidate oligomer (MORF) for its radiolabeling in vivo and invitro and with a biotin for binding to the clearing agent of avidin. Theradiolabeling of the biotin-antibody-MORF was achieved by binding to theradiolabeled MORF complement (cMORF). In contrast to most clearancesystems reported in the literature, the avidin binding of the antibodyin this design does not compete or interfere with its effector binding.

The trifunctional antibody conjugate of the invention includes afunctionality for effective and complete blood clearance and anotherfunctionality for binding to a later administrated effector (e.g., atherapeutic effector or a biomarker probe) that are together linked tothe targeting moiety (e.g., an antibody). For example in tumortargeting, as schematically illustrated in FIG. 1( a), a clearance group(“A”) plus an amine modified effector-binding agent (“B”) is attached toa tumor-specific targeting agent (“C”), such that when the amine becomeslinked to the tumor targeting agent, each A on the tumor targeting agentis with a function B.

Unlike the previous constructs, illustrated in FIG. 1( b), where A and Bare reacted simultaneously with a targeting agent to form the conjugate,the present invention is designed to ensure that each B is covalentlylinked to an A and vice versa before they concomitantly are conjugatedon to the targeting agent. Thus, there is no possibility that only A oronly B is conjugated to the targeting agent. Even if there is impuritythat is not modified with amine, each B on the targeting agent is stillwith a function A, because the unmodified A-B does not react with thetargeting agent. Size-exclusion purification can easily remove theunmodified A-B.

While not wishing to be bound by the theory presented herein, anexplanation for the persistent low clearance of existing targeting andpreclearance strategies is the formation of a product mixture, forexample, in a statistical distribution. FIG. 2A schematicallyillustrates the chemical modification of an antibody by both a biotinand a MORF that may produce a mixture of desired clearable product,undesired non-clearable product, and non-interfering unmodified andpartially unmodified antibody molecules. The product mixture frommodifying an antibody with both NHS-biotin (clearance function) andNHS-SANH plus SFB-MORF (effector-binding function) in the previous artcould produce three different types of products: the desired productwith both biotin and MORF, the undesired by-product with only MORF, andthe unreacted and partially unreacted antibody molecules without MORF.(Liu, et al. 2010 Cancer Biother Radiopharm 25:757-62.) Size-exclusionpurification would not remove the by-product such that the MORF-antibodyinjectate would not be completely clearable.

An exemplary embodiment of a synthetic route according to the inventionis schematically illustrated in FIG. 2B. Biotin and MORF groups areconjugated together to an antibody such that each MORF (aneffector-binding group) was together with a biotin. This uniquetri-functional (avidin-MORF)-antibody pretargeting construct enablescomplete clearance of circulating antibody conjugates. Here, thesynthetic approach employs a mechanism that assures that each MORF onthe antibody co-exists with a biotin.

For example, a 3′-(NH₂-biotin)-MORF was designed, custom-synthesized,and characterized. After confirming each amine was bound to a biotin byradiolabeling with ^(99m)Tc at the amine position and measuring thepercent bound to streptavidin, the (NH₂-biotin)-MORF was conjugated to amodel IgG antibody using a commercial Hydralink method. The MORFimpurity without (NH₂-biotin) modification essentially could not beattached to the antibody and therefore could be removed duringpurification by size exclusion. The (biotin-MORF)-antibody was labeledvia MORF/cMORF-^(99m)Tc hybridization and evaluated in mice for theclearance efficiency by avidin. For a (NH₂-biotin) sample, 75% of theMORF was found with the (NH₂-biotin) modification; however, every aminewas with a biotin. The MORF was readily conjugated to the model antibodyat an average of 1.17 (MORF-biotin)s per antibody. Biodistributions ofthe radiolabeled (biotin-MORF)-antibody with and without avidinindicated that the antibody was almost completely clearable (96% fromblood at 2 and 4 h) and, as expected, the radioactivity was cleared toliver. Thus, by conjugating an amine-plus-biotin modified MORF to anantibody, a completely clearable antibody construct can be madeavailable for future pretargeting applications.

Thus, provided herein is a validated construct for modifying an antibodywith two functions (e.g., MORF and biotin) as a pretargeting agent thatis completely clearable from circulation. Even though the MORF was notpure, each MORF on the antibody in the product was with a biotin becauseonly the (NH₂-biotin)-modified MORF would react with the antibody. Thepretargeting construct obtained in this fashion is tri-functional, i.e.,capable of binding to tumor, clearing agent, and radiolabeled effector.The previous double modification also provided a tri-functional product,but may contain non-clearable impurity because conjugation of twoseparate groups onto an antibody led to a statistical distribution.(Mirallie, et al. 2005 Eur J Nucl Med Mol Imaging 32:901-9; Liu, et al.2010 Cancer Biother Radiopharm 25:757-62.)

Two methods have been employed to measure the number of biotins on theMORF as measures of quality insurance for the above mentioned3′-(NH₂-biotin)-MORF sample. Both indicated that the modification wasincomplete, although the values from the two methods were not identical(0.64 vs 0.75±0.01). Systematic errors from different methods mayreasonably account for the difference. First, quantitation of the MORFby spectrophotometry for the 0.64 biotin per MORF was after hydrolyzingthe MORF by 0.1 N HCl and calculated from the molar absorbance providedby providers, while the HPLC detector was measuring intact MORF. As HPLCmeasures peak area relative to the internal standard, the 0.75±0.01 maybe more accurate. In addition, both methods assumed the MORF sequencewas pure, but the realty would never be so. The peak shape of the MORFthat did not bind to avidin was not identical to that of the total MORF(more easily appreciated if the MORF peaks in FIG. 4B were adjusted tothe same height). Assuming the height was also proportional to theamount as should be in principle, the data led to a larger value of0.84±0.00 biotins per MORF.

In an experiment testing clearablity, avidin and biotinylated antibodywere combined before administering them to animals. It was evident that,if bound to an avidin, a biotinylated antibody clear rapidly andcompletely. This result therefore excludes any possibility that the lowclearance efficiency in the old art was due to a fraction of theavidin-bound antibody still in the circulation. (Liu, et al. 2010 CancerBiother Radiopharm 25:757-62; Liu, et al. 2010 Q J Nucl Med Mol Imaging54:333-40.). Though not directly relating to the theory in thisinvention, the rapid clearance may reduce the risk of immunogenicity ofavidin, a concern naturally arising from the fact that streptavidin wasimmunogenic especially when attached to antibody. (Knox, et al. 2000Clin Cancer Res. 6: 406-14.). Indeed, no immunogenicity for avidin wasever been reported after IV injection probably due to its rapidclearance. (Sinitsyn, et al. 1989 J Nucl Med 30: 66-9.) Subcutaneousinjection did induce immunogenicity, but the period of presence wasprolonged. (Caliceti, et al. 2002 J Controlled Release 83: 97-108.).Thus, immunogenicity for avidin is unlikely to occur if IV injection isused due to rapid clearance.

Thus, in one aspect, the invention generally relates to a conjugatecompound. The compound includes: a targeting moiety capable ofselectively binding to a primary and specific target site or to asubstance produced by or associated with the target site; a firstconjugate moiety comprising two covalently linked ligand groups, a firstligand group and a second ligand group; and a covalent linkage betweenthe targeting moiety and the first conjugate to form a second-orderconjugate compound.

The targeting moiety may be any suitable group dependent on theapplication. In certain embodiments, the targeting moiety is an antibodyor an antibody fragment. Preferred are the monoclonal antibodies (Mabs)due to their high specificities. Mabs may be prepared by procedures ofimmunization of mammals with immunogenic antigen preparation, fusion ofimmune lymph or spleen cells with an immortal myeloma cell line, andisolation of specific hybridoma clones. Other methods of preparingmonoclonal antibodies are also contemplated, such as interspeciesfusions and genetic engineering manipulations of hypervariable regions,since it is primarily the antigen specificity of the antibodies thataffects their utility in the present invention. It will be appreciatedthat newer techniques for production of monoclonals can also be used,e.g., human monoclonals, interspecies monoclonals, chimeric (e.g.,human/mouse) monoclonals, genetically engineered antibodies and thelike.

Useful antibody fragments include, for example, F(ab′)₂, F(ab)₂, Fab′,Fab, Fv and the like including hybrid fragments. Preferred fragments areFab′, F(ab′)₂, Fab, and F(ab)₂. Also useful are any sub-fragmentsretaining the hypervariable, antigen-binding region of an immunoglobulinand having a size similar to or smaller than a Fab′ fragment. Thisincludes genetically-engineered or recombinant antibodies and proteins,whether single-chain or multiple-chain, which incorporate anantigen-binding site and otherwise function in vivo as targetingvehicles in substantially the same way as natural immunoglobulinfragments. (See, e.g., U.S. Pat. No. 4,946,778.) Fab′ fragments may beconveniently made by reductive cleavage of F(ab′)₂ fragments, whichthemselves may be made by pepsin digestion of intact immunoglobulin,under reducing conditions, or by cleavage of F(ab′)₂ fragments whichresult from careful papain digestion of whole immunoglobulin. Thefragments may also be produced by genetic engineering.

In certain preferred embodiments, the antibody or antibody fragment arehumanized. In certain preferred embodiments, the humanized antibody canbe anti-CEA, anti-TAG-72, antibodies.

Other useful antibodies include those having a specific immunoreactivityto a marker substance produced by or associated with the cancer cells ofat least 60% and a cross-reactivity to other antigens or non-targetedsubstances of less than 35%. A monoclonal antibody that specificallytargets tumor sites by binding to antigens produced by or associatedwith the tumors is particularly preferred. Antibodies against tumorantigens are known. For example, antibodies and antibody fragments thatspecifically bind markers produced by or associated with tumors havebeen disclosed. (See, e.g., U.S. Pat. Nos. 3,927,193, 4,331,647,4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709 and 4,624,846.) Inparticular, antibodies against an antigen, e.g., a gastrointestinal,lung, breast, prostate, ovarian, testicular, brain or lymphatic tumor, asarcoma or a melanoma, are advantageously used.

The antibodies and antigen-binding antibody fragments useful in themethods of the present invention may be linked to another group forminga conjugate by a variety of methods of chemical conjugation known in theart. (See, e.g., Childs, et al. 1985 J. Nuc. Med. 26:293.)

In certain embodiments, the targeting moiety is selected from the groupconsisting of proteins, small peptides, polypeptides, enzymes, hormones,steroids, cytokines, neurotransmitters, oligomers, vitamins and receptorbinding molecules.

In certain embodiments, the first ligand group is selected from aMorpholino oligomer, an antibody fragment, or other specific groups.

Morpholino oligomers (“MORFs”) are synthetic molecules that are theproduct of a redesign of natural nucleic acid structure, which couldbind and inactivate selected RNA sequences. But for tumor pretargetingit is selected not binding to RNA. Structurally, the difference betweenMORFs and DNA is that while MORFs have standard nucleic acid bases,those bases are bound to morpholine rings instead of deoxyribose ringsand linked through phosphorodiamidate groups instead of phosphates.(Summerton, et al. 1997 Antisense & Nucleic Acid Drug Development 7 (3):187-95; U.S. Pat. Nos. 5,142,047 and 5,185,444.) MORFs are assembledfrom four monomers, each respectively has one of the four genetic bases(A, G, C, or T), linked to a six-membered morpholine ring.

In certain preferred embodiments, the first ligand group is a Morpholinooligomer (MORF) and the second ligand group is a biotin group. The MORFmay comprise any suitable number of bases, for example, from about 6bases to about 100 bases (e.g., from about 6 bases to about 50 bases,from about 6 bases to about 40 bases, from about 6 bases to about 30bases, from about 10 bases to about 100 bases, from about 10 bases toabout 50 bases). In certain preferred embodiments, the length of theMORF is from about 12 bases to about 30 bases. In certain preferredembodiments, the length of the MORF is from about 15 bases to about 25bases (e.g., 15-mer, 18-mer, 20-mer). In certain preferred embodiments,the length of the MORF is from about 18 bases to about 25 bases. Acomplementary MORF is referred to as cMORF, for example, MORF15 andcMORF15 (15-mer), MORF18 and cMORF18 (18-mer) or MORF25 and cMORF25(25-mer) are complimentary binding pairs.

In another aspect, the invention generally relates to a method fortargeted delivery of an agent to a target site in a mammal. The methodincludes: (a) administering to the mammal a first compound. The firstcompound includes: a targeting moiety capable of selectively binding toa primary and specific target site or to a substance produced by orassociated with the target site, a first conjugate comprising twocovalently linked ligand groups, a first ligand group and a secondligand group, and a second conjugate formed by a covalently linkagebetween the targeting moiety and the first conjugate; (b) administeringto the mammal a clearing agent comprising a group capable of selectivebinding to the second ligand; and (c) administering to the mammal aneffector conjugate, wherein the effector conjugate comprises an agentcovalently linked to a group capable of selective binding to the firstligand group.

The effector conjugate to be delivered to the target site may contain atherapeutic entity or a diagnostic probe. Exemplary therapeutic entitiesinclude antibodies, antibody fragments, drugs, toxins, nucleases,hormones, immunomodulators, chelators, boron compounds, photoactiveagents or dyes and radionuclides. Exemplary diagnostic probes includeradionuclides, dyes, contrast agents, fluorescent compounds ormolecules.

In certain preferred embodiments, the second ligand group is a biotingroup along with a MORF as the first ligand group.

It is noted that any suitable clearing agents may be used in accordancewith the present invention. For example, biotin or streptavidin may beused as the second ligand group in a clearing agent.

Any suitable routes of administration may be employed, for example, byintravenous, intraarterial, intrapleural, intraperitoneal, intrathecal,subcutaneous or perfusion administration.

The method of the invention may be utilized for detection or treatmentof a variety of diseases and conditions, for example, tumors or otherlesions, infectious diseases, inflammatory diseases and autoimmunediseases as well as human pancreatic islets in connection with diabetes.

In yet another aspect, the invention generally relates to a kit fordelivering a diagnostic or therapeutic agent to a target site. The kitincludes: (a) a compound that include: a targeting moiety capable ofselectively binding to a primary and specific target site or to asubstance produced by or associated with the target site, a firstconjugate comprising two covalently linked ligand groups, a first ligandgroup and a second ligand group, and a covalent linkage between thetargeting moiety and the first conjugate; (b) a clearing agentcomprising a group capable of selective binding to the second ligandgroup; and (c) an effector conjugate, wherein the effector conjugatecomprises an effecting group covalently linked to a group capable ofselective binding to the first ligand group.

Examples

A MORF was designed that carries a group containing both biotin and aprimary amine at 3′-end with its sequence the same as used previouslyreported (TCTTCTACTTCACAACTA). (Liu G, et al. 2004 Eur J Nucl Med 31:417-24.). Hereafter in this manuscript, (NH₂-Biotin)-MORF is used forthis amine-plus-biotin modified MORF to indicate both amine and biotinare attached to the MORF. The two functional entities in one group areput in a bracket and smaller group precedes the larger sequentially.Other similar structures will also be expressed following the same rule.

The TEZ™ Biotin Quantitation Kit, streptavidin, and avidin were fromPierce (Thermo Fisher Scientific, Rockford, Ill.), while thestreptavidin agarose sedimented bead suspension was from LifeTechnologies™ (Carlsbad, Calif.). The model antibody CC49 was preparedby Strategic Biosolutions (Ramona, Calif.) from the CC49 murinehybridoma cell line (a gift from Dr Jeff Schlom, Center for CancerResearch, NCI, NIH). Modification of the antibody with the(NH₂-Biotin)-MORF utilized a Hydralink kit consisting of paired agentsof C6-SANH (a succinimidyl-activated hydrazinonicotinate acetonehydrazone) and C6-SFB (a succinimidylactivated formylbenzoate) fromSolulink Biosciences (San Diego, Calif.). All other chemicals werereagent grade and used without purification.

The concentrations of MORF and cMORF were determined by UVspectrophotometry using the molar absorbance from the provider. Sizeexclusion (SE) HPLC was used for their analysis. The HPLC system wasequipped with a superpose-12 10/30 GL column (from GE HealthcareBio-Sciences AB, Uppsala, Sweden, optimal separation range: 1×10³ to3×10⁵ Da) or a Superdex™ 75 column (optimal separation range: 1×10² to7×10³Da; Amersham Pharmacia Biotech, Piscataway, N.J.), a UV in-linedetector, and a radioactivity in-line detector. The eluant of 0.10 M pH7.2 phosphate buffer was commonly used at a flow rate of 0.60 mL/min,unless the 10% ACN-0.10 M NH₄Cl solution was required forstreptavidin/biotin binding studies. Radioactivity recovery wasroutinely measured and was always greater than 90%.

The experimental design for constructing and validating the antibodyconjugate compound is as depicted in the flow chart of FIG. 3. Firstmeasured was the average number of biotin on the MORF and then, toconfirm each amine was with a biotin, the amine on the MORF wasradiolabeled. With this radiolabeled biotin-MORF, it was also determinedwhether avidin could clear the labeled (NH₂-biotin)-MORF into liverprior to conjugating it onto a model antibody (an antiTAG-72 antitumorantibody CC49). After synthesis of the (biotin-MORF)-CC49, the numbersof MORF and biotin per antibody were measured. Finally, it was evaluatedthe clearance efficiency of (biotin-MORF)-CC49 by avidin. Adding^(99m)Tc-cMORF to bind to the pendent MORF creates a label to theantibody.

In a second investigation, the clearance efficiency was investigated todifferentiate the influences of each factor in a pretargeting protocol,including the non-biotinylated MORF-antibody, the long pretargetingtime, and the accessibility of antibody to the avidin.

One study was designed to figure out why a small portion of(biotin-MORF)-CC49 (4%) was non-clearable. The linkage between theantibody and the (NH₂-biotin)-MORF was formed by the reaction of the NH₂group to the NHS-ester linker. We suspect the unmodified impurity mayreact with the NHS ester to a very less extent although theoreticallynot do so. Thus, a NHS-MAG₃ that was able to be labeled afterconjugation was used as a model NHS-ester to investigate the reaction ofNHS-ester with the modified MORFs.

Number of Biotin on (NH₂-Biotin)-MORE

The number of biotin on (NH₂-biotin)-MORF (i.e., the efficiency ofterminal modification or the percent of modified MORF in the product)was measured using EZ™ Biotin Quantitation Kit on a spectrophotometer asdescribed previously. (Liu, et al. 2010 Cancer Biother Radiopharm.25:757-62.) In the kit, HABA was bound to avidin and in color, butreplacement by biotin released and decolorized it such that the decreasein optical density was proportional to amount of biotin. The averagebiotins per MORF were the ratio of biotin over MORF concentrations.

The number of biotin was also measured by SE-HPLC after addingstreptavidin in excess to the (NH₂-biotin)-MORF. The MORF without the(NH₂-biotin) modification would not bind to streptavidin and thereforeshould remain at the original position. Inclusion of an internalstandard (o-iodohippuric acid) allowed for quantitating the remainingunbound (unshiftable) MORF. The number of biotin was calculated by(100%—the percent of unshiftable MORF).

The results measuring the biotins on the (NH₂-biotin)-MORF by the EZ™kit method and the HPLC “shifting” method were shown in FIGS. 4A and 4B.The OD in the Kit method decreases linearly with the amount of MORFadded, yielding a value of 0.64 biotins per MORF and suggesting anincomplete modification. The incomplete modification was furtherconfirmed by the HPLC method. As shown, addition of SA to(NH₂-biotin)-MORF at 1:1 results in a low MORF peak but more SA does notfurther reduce the peak, suggesting the remaining molecules lack abiotin group. The percentage bound to streptavidin, i.e., the number ofbiotin, was quantitated using the internal standard as 1−(Peakarea_(MORF/Standard))_(SA:MORF=n:1)(Peakarea_(MORF/Standard))_(SA:MORF=0:1), (n=1, 2, and 3). The average fromthe three samples=0.75±0.01 biotins per MORF.

Co-Existence of Amine with Biotin in (NH₂-Biotin)-MORF

Similarly to “the shifting” of the native (NH₂-biotin)-MORF on HPLC byan amount of streptavidin in excess, the coexistence of the amine withthe biotin was determined shifting the ^(99m)Tc radiolabeled(NH₂-biotin)-MORF at the amine position. The procedures for conjugatingwith MAG₃ and labeling with ^(99m)Tc were identical to those describedfor the NH₂-cMORF previously. (Liu, et al. 2006 Appl Radiat Isot.64:971-978.) The peak of (^(99m)Tc-biotin)-MORF should shift completelyeven in the presence of unmodified MORF if each amine (and therefore^(99m)Tc) on MORF was together with a biotin.

The coexistence was secondly confirmed by streptavidin-coated beads.After washing three small spin column filled with the beads twice with2% bovine serum albumin in PBS buffer, the (^(99m)Tc-biotin)-MORF wasloaded to each followed by washing twice again. The eluate collectionscontained the ^(99m)Tc labeled MORF that was non-biotinylated similarlyto the negative controls of avidin-bound (^(99m)Tc-biotin)-MORF and abiotin-free ^(99m)Tc-MORF (made exactly following previous protocols.(Liu, et al. 2006 Appl Radiat Isot. 64:971-978.)

Size-exclusion purification of MAG₃-conjugated (NH₂-biotin)-MORF, i.e.,(MAG₃-biotin)-MORF, may contain non-modified MORF impurities. However,the radiolabeled (MAG₃-biotin)-MORF is shifted reasonably completely bythe cMORF as shown in FIG. 5A. This indicated that the specific bindingof the MORF is preserved instead of contradicting to the incompletemodification because the unmodified MORF does not carry a label tocontribute. FIG. 5B further showed that all of the(^(99m)Tc-biotin)-MORF is almost completely shifted by SA (96%),suggesting that the amine site is with a biotin. FIG. 5C confirms thisresult by the fact that the (^(99m)Tc-biotin)-MORF will bind to thestreptavidin beads unlike the biotin-free control ^(99m)Tc-MORF or theavidin-blocked (^(99m)Tc-biotin)-MORF.

Conjugation of (NH₂-Biotin)-MORF to a Model Antibody

The conjugation of (NH₂-biotin)-MORF to an antibody using the commercialHydralink approach was the same as that for the NH₂-MORF. (He, et al.2007 Bioconju Chem. 18: 983-988; Liu, et al. 2010 Q J Nucl Med MolImaging 54:333-40.) Briefly, a model antibody of CC49 and the MORF wererespectively conjugated with C6-SANH and C6-SFB followed bysize-exclusion purification. Combination of the SANH-modified antibodyand the SFB-modified MORF formed a hydrazone link between the antibodyand MORF as shown below. A 1×50 cm Sephadex G-100 glass Econo-column wasused for the purification of the MORF-antibody. The unmodified MORFimpurity, if any, could be removed along with the unreacted(NH₂-biotin)-MORF. The average biotins and MORFs per antibody weredetermined as described previously. (Liu, et al. 2010 Cancer BiotherRadiopharm. 25:757-62.)

Clearance of (Biotin-MORF)-Antibody by Avidin

To avoid unnecessary efforts and to facilitate later interpretation ofthe antibody data, prior to synthesizing the (biotin-MORF)-antibody, theavidin-bound (^(99m)Tc-biotin)-MORF was administered to mice. Afterconfirming the avidin clearance effect and subsequent success inconjugating (NH₂-biotin-MORF) to antibody, the antibody construct waslabeled by adding tracer amount of ^(99m)Tc-cMORF and evaluated for theavidin clearance. The ^(99m)Tc-cMORF was obtained from instant labelingof a MAG₃-MORF stock solution. (Liu, et al. 2006 Appl Radiat Isot.64:971-978.) The clearance studies by avidin included two injectates forin each case: an avidin-added for clearance and the other without avidinas a negative control. All animal use was in accordance with theguidelines of the Animal Care and Use Committee of the University ofMassachusetts Medical School and conformed to the recommendations in theGuide for the Care and Use of Laboratory Animals (Institute ofLaboratory Animal Resources, National Research Council, National Academyof Sciences, 1996).

In animals for testing the antibody construct, an injectate containing30 μg of (biotin-CC49)-MORF/cMORF-^(99m)Tc (43 μCi, 1.17 MORF-biotinsper CC49, MORF/cMORF ratio=2) or 30 μg of the antibody complex plus 80μg of avidin (avidin/MORF or avidin/biotin=5) was administered to eachof 20 CD-1 mice. They were divided into 5 groups (N=4) and euthanized at2 min, 30 min, 1 h, 2 h, and 4 h. Organs were removed, weighed, andcounted in an autogamma counter along with the injectate standardsfollowing our routine procedure and the % ID and % ID/g values werecalculated. (Liu G, et al. 2004 Eur J Nucl Med 31: 417-24.) For the(^(99m)Tc-biotin)-MORF, 1.0 μg (50 μCi) was administrated and theavidin/biotin ratio in the study injectate was also 5. Six groups of 4mice were used and euthanized within a shorter period at 2 min, 12 min,30 min, 1 h, 2 h, and 3 h, because the small MORF not attaching to anantibody cleared much faster.

Organs of especial interests for evaluating the clearance efficacy byavidin include blood, to observe how fast a biotinylated agent leavesthe circulation; liver, to confirm whether avidin clears thebiotinylated agents into this organ; whole body, to determine how muchof the agent is completely out; and kidney, particularly relating to thelabeled MORF oligomer, to check whether the clearance pathway has beenaltered.

As shown in FIG. 6A, the (^(99m)Tc-biotin)-MORF clears rapidly fromblood just like the previous biotin-free ^(99m)Tc-MORF, but afterbinding to avidin, the blood clearance is further accelerated. Theavidin effect can be appreciated more easily from the liver leveldifference, an accumulation of 55% ID with avidin at 3 h at 172-folds ofthat without avidin (0.32% ID). The free (^(99m)Tc-biotin)-MORF leavesthe body through kidneys rapidly with only ˜13% ID left in the body at 3h (excluding bladder). Almost nowhere retains except kidneys (10% ID).When bound to avidin, the (^(99m)Tc-biotin)-MORF remains in the wholebody although rapidly leaves the circulation. Its kidney accumulation istwice higher than that of the free (^(99m)Tc-biotin)-MORF but notexcreting through this organ.

The (biotin-MORF)-antibody/cMORF-^(99m)Tc is much bigger in sizecompared to the (^(99m)Tc-biotin)-MORF (160 kDa vs 6 kDa), leading to astriking difference in clearance rate from blood or circulation as shownin FIGS. 6A and 6B. Therefore, the avidin effect becomes prominent evenin the blood. The clearance efficiency at 4 h is at(30−1.2)/30*100%=96%, in coincidence with the 96% streptavidin-shiftable(^(99m)Tc-biotin)-MORF. Avidin clearance in blood takes about 1 hcompared to the almost immediate for the (^(99m)Tc-biotin)-MORF. Alsobecause of the bigger size, consistent to normal radiolabeledantibodies, it gets into liver by itself, but after bound to avidin, itclears to liver at a much higher rate and greater percentage as comparedto the (^(99m)Tc-biotin)-MORF. (Cansow 1991 Nucl Med Biol 18: 369-381.)Again because of the bigger size, either bound to avidin or not, thelabeled antibody construct does not excrete through kidneys and not evenaccumulate there (only 1.4-1.8% ID). Irrespective of the avidin-boundstatus, the whole body retention is identically high within theobservation period, because the label on the antibody accumulated inliver either naturally or by avidin excretes slowly.

Clearance Efficiency by a Clearance Step with Varying PretargetingInterval

Because a longer wait also reduces the non-specific background,pretargeting interval and avidin clearance as to their clearance effectswere examined. The effect of avidin (clearance efficiency) is defined asthe percentage of blood concentration reduction by avidin clearance ascompared to the control without avidin clearance. Thebiotin-antibody-MORF was prepared from the old art with more than 12% isnot clearable. This experiment was conducted to show the state of theMORF-antibody in blood. To completely label the antibody in vivo, thecMORF amount is in a molar excess of many folds as compared to the MORFon the antibody, such that the blood level of the cMORF is proportionalto that of the antibody and can be used as a measure of the antibodyblood level. The relationship is: % ID/g_(antibody)=%ID/g_(cMORF)/(Molar dose ratio of cMORF/antibody×MORF number perantibody). We note the actual antibody level is higher than the cMORFlevel because only a very small portion of the rapidly-clearing cMORF isconsumed for binding to the MORF-antibody.

In a study set of 3 groups of mice (N=4), each of the 12 mice receivedvia tail vein 30 μg of biotin-CC49-MORF (0.67 MORFs and 3.41 biotins perantibody); 1, 2, or 3 days later, an avidin dose of 34 μg wasadministrated intravenously to each of the 4 mice of the 3 groupsfollowed 3 h later by the injection of 1.2 μg (80 μCi) of^(99m)Tc-cMORF. The mice were euthanized for biodistribution at 3 hsubsequent to the radioactivity injection. In a control study, anotherwise identical procedure was applied except for the removal of theavidin injection. The 1.2 μg of cMORF was an optimized mass dose thatwas sufficient to saturate the MORF-antibody and the 34 μg of avidin waspreviously determined to be more than sufficient to saturate thebiotin-modified MORF-antibodies in the circulation. (Liu, et al. 2010Cancer Biother. Radiopharm. 25, 757-762.) The decrease of bloodradioactivity level was proportional to the decease of the MORF-antibodylevel and was used as the measure of background improvement. Theclearance efficiency (=100%−the ratio of the blood levels ofradioactivity with or without avidin injection) was used to quantify theclearance effect of avidin.

Both prolonging the pretargeting interval and the use of avidin reducedthe antibody levels in blood (FIG. 7) and normal tissues (notpresented), but the use of avidin is more effective. The clearanceefficiency using avidin as a clearing agent is 55-58%, i.e., with >40%antibody remaining in the circulation. The MORF-antibody moleculescleared from circulation and the remaining seem equally clearable,because varying the pretargeting interval did not considerably changethe clearance efficiency (FIG. 7).

As confirmed in FIG. 8, the additional injections of 33.4 μg of avidinto the pretargeted animals subsequent to the first further reduce theblood concentration of antibody. This suggests that the majority ofantibody molecules that survived the first avidin injection areclearable, consistent with the expectation from the 88% of the clearableavidin-treated antibody measured using the method disclosed hereinabove. The collective clearance efficiency following 3 avidin injectionsis 80% (0.61/3.10*100%). The separate clearance efficiency followingeach injection is 43% (1-1.78/3.10), 44% (1-0.99/1.78), and 38%(1-0.61/0.99) respectively. The real clearance efficiency may beactually higher because of the non-clearable portion. A calculationbased on a constant clearance efficiency of 51%, and the non-clearableportion of 12% provides the blood concentrations of 3.10, 1.71, 1.03,0.69% ID/g. These numbers are in a good agreement to the measured3.10±0.24, 1.78±0.33, 0.99±0.07, 0.61±0.05% ID/g.

The in vivo interaction between antibody and clearing agent has becomeclear now. The results should be useful for both immunotherapy usingantibody-drug conjugates as well as for immunoradiotherapy usingpretargeting. We demonstrated the every avidin bound antibody moleculein blood leaves the circulation very rapidly, differentiated thetemporarily non-clearable and the real non-clearable antibody, andturned the previous hypotheses into facts. The multiple injection studyconfirms the temporary on-clearable concept and supports that acontinuous administration (infusion) of the clearing agents generates acomplete clearance of the clearable MORF-antibody and is suitable forthe future clinical applications.

Reaction of NHS-Activated Ester with Primary Amine-Derivatized DNAAnalogue and Non-Derivatized Impurity

This experiment was designed to figure out why the 4% of(biotin-MORF)-CC49 was non-clearable. The linkage between the antibodyand the (NH₂-biotin)-MORF was formed by the reaction of the NH₂ group tothe NHS-ester linker. We suspect the unmodified impurity may react withthe NHS ester to a very less extent although theoretically not do so.Thus, a NHS-MAG₃ that was able to be labeled after conjugation was usedas a model NHS-ester to investigate the reaction of NHS-ester with themodified MORFs. As shown in FIG. 9, the amine in the modified MORF is aprimary amine and terminal of the un-modified amine is a secondaryamine. We conjugated a mixture of an amine-derivatized MORF and a nativeMORF with NHS-MAG₃ and radiolabeled the conjugates for investigation. Inthe primary amine-derivatized MORF, the biotin concomitantly with theprimary amine was used for streptavidin (SA)-mediated discriminationbetween the conjugate of (NH₂-Biotin)-MORF and that of non-derivatized(or native) MORF. If the labeled MORF did not bind to SA, then theMAG₃-MORF was formed by side reactions instead of by reacting to theprimary amine.

The MORF and its complement (cMORF) were custom-synthesized byGene-Tools (Philomath, Oreg.). The MORF was also derivatized byattaching a primary amine-plus-biotin group to the 3′-equivalentterminus by the company. The MORF sequence was (5′-TCTTCTACTTCACAACTA)and the terminal structures of the two MORFs are illustrated in FIG. 9.The S-acetyl NHS-MAG₃ was previously synthesized in house. (Liu, et al.2002 Bioconjug. Chem. 13: 893-897.) The EZ™ Biotin Quantitation Kit andthe streptavidin were from Pierce (Thermo Fisher Scientific, Rockford,Ill.). The P-4 resin (Bio-Gel P-4 Gel, medium) was from Bio-RadLaboratories (Hercules, Calif.). The ⁹⁹Mo—^(99m)Tc generator was fromPerkin Elmer Life Science Inc (Boston, Mass.). All other chemicals werereagent grade and were used without purification.

As shown in FIG. 10, the (NH₂-biotin)-MORF, the native MORF control, anda 50/50 mixture of the two were treated with the S-acetyl NHS-MAG₃following a reported protocol. (Liu, et al. 2006 Appl. Radiat. Isot. 64:971-978.) 1.5 mg of MORF was dissolved in 0.2 M, pH 8.0 HEPES to aconcentration of 0.5 mg/mL. The solution was added to a vial containingS-acetyl NHS-MAG₃ such that the MAG₃/MORF molar ratio was 20. After 2 hincubation at room temperature, the reaction mixture was purified over aP4 column (0.7×20 cm) using 0.25 M NH₄OAc at pH 5.2 as eluent. The peakfractions with optical density (OD) value at 265 nm greater than 5 werepooled. Subsequently, a procedure was performed to dissociate theunstable MAG₃-MORF conjugate. The MORF solution was mixed with atartrate buffer (pH 9.2, 50 μg/μL Na₂Tartrate.2H₂O in a solution of 0.5M Na₂HCO₃, 0.25 M NH₄OAc, and 0.175 M NH₃) and a fresh tin solution (10μg/μL SnCl₂.2H₂O and 1 μg/μL NaAscorbate in 10 mM HCl). The volumeratios of MORF solution/tartrate buffer/tin solution were 15/5/1. Themixed solution was heated at 100° C. for 20 min and purified again overa longer P4 column (1.0×50 cm), followed by pooling the peak fractionswith OD values over 5 as the final stock conjugate solution.

Each MAG₃-conjugated MORF solution was labeled with ^(99m)Tc. Between 5and 50 μL of ^(99m)Tc-pertechnetate generator eluate was added to amixed solution of 30 μL of MAG₃-MORF solution (0.2-0.4 mg/mL MORF) in pH5.2 NH₄OAc buffer, 10 μL of tartrate buffer, and 3 μL of 4 μg/μLSnCl₂.2H₂O in ascorbate-HCl solution (1 μg/μL NaAscorbate in 10 mM HCl),followed by heating at 100° C. for 20 min.

To confirm that the radioactivity peak did represent faithfully thelabeled MORFs, they were hybridized with cMORF. When the duplex formed,the labeled single-strand radioactivity peak shifted to the position ofthe duplex of doubled molecular weight. The cMORF was in a great excess(at a cMORF/MORF molar ratio of ˜55) as compared to the labeled MORF.Thus, the cMORF were more than sufficient to bind any of the MORFpreserving the hybridizing affinity.

The labeled MORFs were also reacted with SA such that any labeled MORFmolecule that carried a biotin was shifted to the SA position orslightly further to the left. An excessive dose resulting in a molarratio of SA/MORF=10:1 was used to assure a complete shift of thebiotinylated MORF.

The results from the detection by the EZ™ kit and from the HPLC“shifting” are shown in FIGS. 11A and 11B. The OD value from the kitmethod decreases linearly with increasing MORF added. The number ofbiotins per MORF is calculated to be 0.68 by the formula of(slope*MW*volume)/(34000*MORF concentration), a value of lower than 1indicating an incomplete terminal derivatization. This result isconsistent with the results from the HPLC method as shown in FIG. 11B.Addition of streptavidin to (NH₂-biotin)-MORF at a molar ratio of 1:1“shifts” most of the MORF peak to the left. Further addition ofstreptavidin has no effect, confirming the residual MORF lacks the(NH₂-biotin). The fraction of residual peak area after addition ofstreptavidin was calculated to be 0.65±0.01 based on the formula of[1−residual peak area_(n:1)]/[original peak area_(0:1)], (n=1, 2 and 3).

As shown in the top row of FIG. 12, after MAG₃ modification, both the(NH₂-biotin)-MORF and the native MORF can be labeled with highefficiency (>95%), confirming our suspicion. All can hybridize with thecMORF (2^(nd) row), indicating the overall integrity of the sequence.The fact that the native MORF can be labeled indicates the NHS-esteralso reacts with other groups in addition to the primary amine.

Nevertheless, the SA shifting indicates the (NH₂-biotin)-derivatizedMORF reacts with the NHS ester in a much higher yield than thenon-derivatized MORF. Certainly, the ^(99m)Tc-MAG₃ labeled native MORFcannot be shifted by SA due to the lack of biotin. Most of the^(99m)Tc-MAG₃ labeled (NH₂-biotin)-MORF is shifted (˜95%), reproducingour earlier results with another (NH₂-biotin)-MORF sample. Thereactivity of the non-derivatized MORF with NHS-MAG₃ relative to that ofthe (NH₂-biotin)-derivatized MORF can be estimated theoretically, butthe ˜5% unshiftable residual is too small for an accurate estimation,which was the 1:1 mixture of (NH₂-biotin)-MORF and native MORF wasincluded. Assuming the 35% of the (NH₂-biotin)-MORF sample is in thenative form of the MORF (based on the absence of biotin and the personalcommunication with Dr Yongfu Li from Gene-Tools), the 70.7% shift forlabeled mixture (middle of the bottom row) indicates that 5 molecules of(NH₂-biotin)-MORF reacted with the NHS ester for each of the native MORFmolecules that reacted with the NHS ester. In other words, thereactivity of native MORF is 5 times weaker than the (NH₂-biotin)-MORFunder the conditions of this investigation.

Referring to FIG. 9, the reaction sites on the native MORF for theNHS-ester possibly include the 3′-primary amine and the aromatic aminesin the residual bases. Those in the (NH₂-biotin)-MORF are the sameexcept the 3′-terminal secondary amine is converted to a primary amine.The primary amine is much more active site than the secondary amine. Asdepicted in FIG. 13, the MAG₃ conjugation to the native MORF can beattributed to the secondary amine and others, although we do not knowtheir exact relative contributions. For the (NH₂-biotin)-MORF, wesimilarly do not know the contribution of the other reaction sites thanthe terminal amine. Nevertheless, as seen in FIG. 13, we do know the4-fold increase in reactivity is due to the primary amine, because thereactivity difference between native MORF and (NH₂-biotin)-MORF comesfrom the conversion of the secondary amine to the primary amine. Inaddition, the contribution from the secondary amine for the native MORFshould now be credited to the primary amine, because the other possiblereaction sites are identical. Thus, although the native MORF can beconjugated and provides an equally high labeling efficiency, the primaryamine attached to the (NH₂-biotin)-MORF accounts for at least 80% of theentire MAG₃ conjugation. In reality, it may be closer to 90 or 95% asthe secondary amine is more reactive than aromatic amines.

The above results apparently contradict with our previous assumptionthat the impurity of MORF would not interfere with the clearability ofbiotin-Antibody-MORF sample. Nevertheless, in addition to that the 4%biotin-Antibody-MORF sample can be now explained, it provides chemicalevidence that the previous assumption is essentially reliable.Furthermore, if the (NH₂-biotin)-MORF is pure, our design will give riseto a completely clearable antibody conjugate.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is:
 1. A conjugate compound comprising: a targetingmoiety capable of selectively binding to a primary and specific targetsite or to a substance produced by or associated with the target site; afirst conjugate moiety comprising two covalently linked ligand groups, afirst ligand group and a second ligand group; and a covalent linkagebetween the targeting moiety and the first conjugate to form asecond-order conjugate compound.
 2. The compound of claim 1, wherein thefirst ligand group is selected from Morpholino oligomers, peptides,toxins, enzyme, hormones, immunomodulators, chelators, photoactiveagents, dyes, optical probes, and radionuclides.
 3. The compound ofclaim 1, wherein the targeting moiety is an antibody or an antibodyfragment.
 4. The compound of claim 3, wherein the antibody or antibodyfragment are humanized.
 5. The compound of claim 1, wherein thetargeting moiety is selected from the group consisting of proteins,small peptides, polypeptides, enzymes, hormones, steroids, cytokines,neurotransmitters, oligomers, vitamins and receptor binding molecules.6. The compound of claim 2, wherein the length of the Morpholinooligomer is from about 6 bases to about 100 bases.
 7. The compound ofclaim 6, wherein the length of the Morpholino oligomer is from about 10bases to about 50 bases.
 8. A kit for delivering a diagnostic ortherapeutic agent to a target site, the kit comprising: a compoundcomprising: a targeting moiety capable of selectively binding to aprimary and specific target site or to a substance produced by orassociated with the target site, a first conjugate comprising twocovalently linked ligand groups, a first ligand group and a secondligand group, and a covalent linkage between the targeting moiety andthe first conjugate; a clearing agent comprising a group capable ofselective binding to the second ligand group; and an effector conjugate,wherein the effector conjugate comprises an effecting group covalentlylinked to a group capable of selective binding to the first ligandgroup.
 9. The kit of claim 8, wherein the first ligand group is aMorpholino oligomer and the second ligand group is a biotin group. 10.The kit of claim 8, wherein the agent is a therapeutic agent.
 11. Thekit of claim 8, wherein the agent is a diagnostic agent.
 12. The kit ofclaim 10, wherein the therapeutic agent is selected from the groupconsisting of antibodies, antibody fragments, drugs, toxins, nucleases,hormones, immunomodulators, chelators, boron compounds, photoactiveagents or dyes and radionuclides.
 13. The kit of claim 11, wherein thediagnostic agent is selected from the group consisting of radionuclides,dyes, contrast agents, fluorescent compounds or molecules.
 14. The kitof claim 8, wherein administration is by intravenous, intraarterial,intrapleural, intraperitoneal, intrathecal, subcutaneous or perfusionadministration.
 15. The kit of claim 8, used for internal detection ortreatment of tumors or other lesions, infectious diseases, inflammatorydiseases, and autoimmune diseases.
 16. The kit of claim 15, wherein thetargeting moiety is a tumor-specific antibody or an antibody fragment.17. The kit of claim 16, wherein the antibody or antibody fragment arehumanized.
 18. The kit of claim 9, wherein the length of the Morpholinooligomer is from about 6 bases to about 100 bases.
 19. The kit of claim8, wherein the target site are pancreatic islets.
 20. The kit of claim19, wherein the pancreatic islets are imaged in diagnosis of diabetes.